SOURCES OF CALCIUM UTILIZED IN CHOLINERGIC RESPONSES IN CANINE COLONIC SMOOTH-MUSCLE

Ratiometric fura 2 fluorescence techniques were used to investigate the sources of Ca2+ that lead to an increase in cytosolic Ca2+ concentration ([Ca2+](i)) and the generation of force during cholinergic stimulation of canine colonic circular smooth muscle tissues. Acetylcholine (ACh; 1 mu M) caused a biphasic increase in [Ca2+](i) and force. The initial upstroke phase was characterized by an increase in [Ca2+](i) and a pronounced increase in force. The sustained phase was characterized by concurrent oscillations in [Ca2+](i) and force (2-3/ min) that persisted as long as ACh was present. The increase in [Ca2+](i) in response to ACh was reduced to similar to 30% in the presence of nicardipine (1 mu M), suggesting that L-type Ca2+ channels contribute to the rise in [Ca2+](i) but that other sources also contribute. Preincubation in caffeine (10 mM) and ryanodine (10 mu M) reduced the upstroke phase of the increase in [Ca2+](i) and contractile responses to ACh, indicating that release of Ca2+ from intracellular stores contributes only to the initial cholinergic response. Responses to ACh persisted when nicardipine (1 mu M) was included after emptying of caffeine-ryanodine-sensitive stores, suggesting the presence of additional sources of Ca2+. Data suggest that cholinergic regulation of [Ca2+](i) in colonic smooth muscle occurs by a number of parallel pathways. Influx through voltage-dependent Ca2+ channels, release of Ca2+ from intracellular stores, and possibly Ca2+ entry through additional conductances activated by ACh all contribute to the regulation of [Ca2+](i).